verbatim): This proposal is designed to test hypotheses related to gene transfer of adenylyl cyclases to modulate agonist-specific cyclic AMP generation of cardiovascular cells. Adenylyl cyclase (AC) expression appears to be the limiting component for maximal generation of cyclic AMP by agonists, such as beta-adrenergic agonists, that promote such generation. Our preliminary data in cardiac myocytes demonstrate that increased expression of AC-6 (one of the ten isoforms of this enzyme) dramatically enhances beta-adrenergic response without enhancing response to several other stimulatory agonists but while retaining and perhaps enhancing ability of the muscarinic cholinergic agonist carbachol and endothelin to inhibit cAMP generation. We propose to test the impact of increasing expression of AC-6 (using an adenoviral construct) on cyclic AMP formation in key cardiovascular cells, endothelial cells, and vascular smooth muscle cells, in addition to cardiac myocytes, and in particular, to characterize the patterns of stimulation and inhibition of AC-6 in such cells. In other Aims we will test hypotheses developed to explain the selective enhancement of beta-adrenergic response by overexpressed AC-6: 1) preferential coupling of beta-adrenergic receptors to AC-6 compared to certain other AC isoforms (by testing impact of adenoviral transfer of AC-4 and AC-8, each with its own unique pattern of regulation, to cardiac cells); 2) inhibitory regulation of AC-6 that may contribute to patterns of receptor stimulation of cAMP synthesis; and 3) targeting of AC isoforms to membrane domains enriched in certain receptors and other components that regulate cyclic AMP formation and action. Results of these studies should yield new information regarding the role of AC expression as a limiting component in cAMP formation, the impact of gene transfer of AC isoforms to several key cardiovascular cells, and provide evidence that this approach may provide a novel means to alter signaling in such cells.
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